TGF-β-like proteins, i.e. proteins of the TGF-β superfamily play a central role in many biological regulation pathways such as embryonal development or regeneration of tissue. They are very potent biological agents which can be used also therapeutically for a series of different purposes. The best known members of the TGF-β superfamily are the TGF-βs themselves.
TGF-β was originally purified to homogeneity from human platelets, human placenta and bovine kidney and identified as a homodimeric protein with a molecular mass of about 25.000 Da. First characterized by its ability to act synergistically with EGF or TGF-α to induce anchorage-independent growth of untransformed NRK cells, recently, TGF-β has been shown to exhibit numerous regulatory effects on a wide variety of both normal and neoplastic cells indicating the importance of this protein as a multifunctional regulator of cellular activity. Depending upon the cell or tissue type, and the presence or absence of other growth factors, TGF-β may either stimulate mitogenesis, cell proliferation and growth, or may effectively inhibit said processes, or may exhibit other actions like e.g. control of adipogenesis, myogenesis, chondrogenesis, osteogenesis und immune cell function, stimulation of chemotaxis, or induction or inhibition of differentiation. Many of the actions of TGF-β are related to the response of cells or tissues to stress or injury, and to the repair of resultant damage. After inflammation, TGF-β plays the major role in the formation of granulation tissue, increases the expression of genes associated with extracellular matrix formation such as fibronectin, collagen and several protease inhibitors and stimulates collagen-matrix contraction by fibroblasts, suggesting its possible role in connective tissue contraction.
Until now five distinct, however, functionally and structurally closely related TGF-βs designated as TGF-β1, TGF-β2, TGF-β3, TGF-β4 and TGF-β5 are described. The former three are also found in man.
All TGF-βs are synthesized as 390 to 412 amino acid precursors that undergo proteolytic cleavage to produce the mature forms, which consist of the C-terminal 112 amino acids. In their mature, biologically active forms, TGF-βs are acid- and heat-stable disulfide-linked homodimers of two polypeptide chains of 112 amino acids each. The complete amino acid sequences of human (Derynck, R. et al. (1985) Nature 316, 701–705), murine (Derynck, R. et al. (1986) J. Biol. Chem. 261, 4377–4379) and simian TGF-β1 (Sharples, K. et al. (1987) DNA 6, 239–244) show remarkable sequence conservation, differing only in a single amino acid residue. Comparison of the amino acid sequence of human TGF-β1, human TGF-β2 (deMartin, R. et al. (1987) EMBO J. 6, 3673–3677; Marquardt, H. et al. (1987) J. Biol. Chem. 262, 12127–12131) and human TGF-β3 (Ten Dijke, P. et al. (1988) PNAS 85, 4715–4719) has demonstrated that the three proteins exhibit in their mature forms about 70–80% sequence identity. A heterodimeric TGF-β1.2 has been isolated from porcine platelets and consists of one subunit of TGF-β1 disulfide-linked to one subunit of TGF-β2 (Cheifetz, S. et al. (1987) Cell 48, 409–415).
Recently, attempts have been undertaken aiming to produce TGF-βs by means of recombinant techniques rather than isolating these factors from natural sources (e.g. platelets) in order to obtain sufficient amounts for testing in various therapeutic modalities. However, it has proven to be extremely difficult to obtain biologically active recombinant TGF-β. As can be seen from the sequences depicted in the sequence listing under SEQIDNos.1 to 6, the 112 amino acids containing mature forms of TGF-β1, TGF-β2 and TGF-β3 contain 9 cysteine residues. As has been shown for TGF-β2 the 9 cysteine residues are forming 4 intrachain and 1 interchain disulfide bonds [Schlunegger, M. P. and Gruetter, M. G., Nature 358:430–434(1992)]. Heterologous expression of TGF-β may lead to a product which, although having the correct primary structure, fails to fold properly to produce the correct secondary or tertiary structures and which, therefore, lacks the biological activity.
Taking the complexity of the native TGF-β molecules into account, it has generally been considered expedient to express the respective TGF-β genes in cells derived from higher organisms. Although expression of recombinant TGF-βs can be achieved in eukaryotic systems, the yields of biologically active, correctly folded material obtained are still far from being satisfactory.
Therefore, attempts were made to produce biologically active TGF-β in a microbial host. However, in e.g. bacteria the intracellular conditions are not conducive to correct folding, disulfide bond formation and disulfide-stabilized dimerization which is apparently essential for activity. Thus, only very little biologically active TGF-β could be obtained after expression of the respective gene in E. coli under the control of the lambda promoter as described in European Patent Application EP-A-0 268 561. Another report describes the expression of a TGF-β cDNA in E.coli under the control of the trp promoter yielding a radioactively labelled protein band with an apparent molecular weight of 13′000 Da in an autoradiogram of a SDS polyacrylamide gel, but no activity was measured (Urushizaki, Y. et al. (1987) Tumor Res. 22, 41–55).
When recombinant proteins are produced at high levels in bacterial (such as E. coli) expression systems, they often appear in the form of highly insoluble intracellular precipitates referred to as inclusion bodies or refractile bodies which can be recognized as bright spots visible within the enclosure of the cells under a phase contrast microscope. These inclusion bodies, which can readily be separated from the soluble bacterial proteins, contain the recombinant protein in a mostly denatured and reduced form which does not exhibit the functional activity of its natural counterpart and which therefore is useless as a commercial product. It is therefore generally agreed, that the recombinant refractile protein has to be solubilized under conditions which are suitable in maintaining it in its denatured form and subsequently has to be folded in order to undergo the transition from the denatured unfolded form to the proper, functionally active three-dimensional structure, the conformation of which is stabilized by relatively weak interatomic forces such as hydrogen bonding, hydrophobic interactions and charge interactions. In the case of cysteine containing proteins this process may also involve formation of disulfide bonds. When the formation of disulfide bonds is chemically promoted, the formation of incorrect intramolecular and, in the case of dimeric or multimeric proteins, intermolecular bridges should be prevented or at least minimized, since the formation of undesired, incorrectly folded isomers may yield non-homogenous material, thus complicating the further purification of the protein having the desired structure, or may generate a protein with reduced activity.
Folding of proteins usually is performed in a multistep process comprising the solubilization of the protein under strongly denaturing conditions, and then reducing the concentration of the chaotrop in order to allow the folding of the protein. However, such an approach failed in the folding of TGF-β. In the European patent application EP-A-0 433 225 a successful process for the production of biologically active, dimeric TGF-β-like protein is described, in which a mild detergent is used which allows the folding of the TGF-β-like protein while the detergent is present in the folding buffer.
It is known from the prior art (Tam et al., J. Am. Chem. Soc. 113:6657–6662, 1991) that dimethyl sulfoxide (DMSO) can be used for promoting a selective and efficient formation of disulfide bonds in peptides. The method is selective, i.e. without side reactions, and a wide pH range can be applied. However, correct disulfide bridge formation was shown only for peptides up to about 30 amino acids. In another publication (Bentle et al., U.S. Pat. No. 4,731,440) dimethylsulfone or a mixture of dimethylsulfone and urea was used for solubilization of somatotropin from inclusion bodies. The solubilized protein then could be renatured by contacting the dimethylsulfone containing solution of the protein with a mild oxidizing agent.
Surprisingly it was now found that TGF-β-like proteins can be refolded into the active, dimeric form by treating the solubilized monomer with a detergent-free folding buffer which comprises an organic solvent such as e.g. DMSO, DMF or a mixture of DMSO and DMF.